Chemical sensing device

ABSTRACT

An apparatus with a transducer having a first output signal and arranged to receive an electrical input. The transducer switches the first output signal between an ON and OFF state. The apparatus has a chemical sensing surface coupled to the transducer arranged to receive a chemical input. A signal generator oscillates one or more of said inputs to vary the switching point of the transducer. The oscillating input may be the chemical input and/or the electrical input. The output signal may be a pulse whose period ON or OFF is determined by the oscillating input modulated by the chemical input.

FIELD OF THE INVENTION

The present invention relates to devices for chemical sensing. Inparticular the invention may be used to provide a digital output signaldependent on the concentration of an ion in a fluid. The invention isapplicable to nucleic acid identification and sequencing.

BACKGROUND

Previously publications have disclosed the ability of an ION SensitiveField Effect Transistor (ISFET) to detect chemicals proximate thesensing surface. This may be used to determine the presence of a targetanalyte by detection of products of a chemical reaction. In one example,ISFETs can be used determine the identity of one or more portions of anucleic acid template by detecting the change in pH resulting fromnucleotide insertion at the end of a nucleic acid. Typically hydrogenions (protons) are released during the reaction. The electrical signalstrength of the ISFET depends on the amount of hydrogen ions released,which is expressed as an analogue output signal, which is either avoltage or current signal.

For large scale arrays of ISFETs, such as might be used in DNAsequencing, the inventors have appreciated that processing this analoguedata requires enormous computing power and a bandwidth ofgigabits/second.

In addition, the normal method requires accurate analogue readoutcircuitry, and is sensitive to the parasitic components, andenvironmental electrical noise. High accuracy and large-scale analoguesystems limit the processing speed and integration ability; therebyconstraining the detection efficiency and scalability. Moreover, highperformance front-end interface circuitry consumes large amounts ofsystem area and power, make on-chip data processing unrealistic.

The inventors propose herein a novel semiconductor and method thataddresses one or more of the above deficiencies.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided asemiconductor device comprising a transducer having a first outputsignal and arranged to receive an electrical input. The transducerswitches the first output signal between an ON and OFF state. The devicecomprises a chemical sensing surface coupled to the transducer arrangedto receive a chemical input. A signal generator oscillates one or moreof said inputs to varies the switching point of the transducer.

The oscillating input may be the chemical input and/or the electricalinput.

The output signal may be a pulse whose period ON or OFF is determined bythe oscillating input modulated by the chemical input.

The signal generator may comprise a titration electrode arranged torelease or adsorb a chemical, which chemical affects a concentration ofions detected by the chemical sensing surface. The signal generator mayfurther comprise a controller to provide an oscillating electricalcurrent to the titration electrode.

Said chemical is preferably hydrogen ions or hydroxide ions.

The signal generator may oscillate the chemical input signal to switchthe first output signal ON for a first period and OFF for a secondperiod. The signal generator may also oscillate the electrical inputsignal to switch the first electrical output signal ON for a firstperiod and OFF for a second period; further wherein the chemical inputsignal modulates the period of the first and second period.

The signal generator may provide an oscillating electrical input signalto a transistor comprised in the transducer at one of: a gate, a source,a drain, or a bulk.

The ions to be detected proximate the chemical sensing surface mayprovide the chemical input signal to bias the transducer to modulate thefirst output signal.

The transducer may comprise a first transistor of a first type connectedto a second transistor of a second type to form a CMOS inverter. Thechemical sensitive surface may be coupled to gates of the first andsecond transistors. The gate may be a floating gate.

The first electrical output signal may be provided to one or morefurther transistors to form a logic circuit.

The apparatus may further comprise a demodulator receiving the firstelectrical output signal, the demodulator providing a second digitaloutput signal representing the magnitude of the chemical input signal.

The demodulator may be one of: a Phase Demodulator or Time to DigitalConvertor (TDC).

The apparatus may further comprise an encoder connected to the secondoutput signal, the encoder arranged to provide a third output signalcomprising a 2-bit binary code.

Preferably, an oscillating waveform of the signal generator is providedas one of: a sawtooth wave, a sinusoidal wave, or a triangular wave.

According to a second aspect of the invention there is provided a methodof determining one or more components of an analyte in a fluid using Anapparatus according the first aspect, and comprising the steps of (i)providing the fluid to the chemical sensing surface; (ii) oscillatingone or more of the inputs to provide a modulated first output signal;(iii) demodulating the modulated first output signal to provide a firstdata representing a first ion concentration; (iv) combining an analytespecific reagent with the fluid, whereby ions are produced if thereagent reacts with the analyte; (v) oscillating one or more of theinputs to provide a modulated first output signal; (vi) demodulating themodulated first output signal to provide a second data representing asecond ion concentration; (vii) comparing the first data and second datato quantify a change in ion concentration; and then (viii) comparing thechange in ion concentration with a threshold to determine whether thereagent reacted with the analyte to determine a component of theanalyte.

The method may further comprise demodulating the first and secondmodulated output signals to provide first and second digital outputsignals representing an ion concentration and then comparing the firstand second digital output signals to quantify a change in ionconcentration of the fluid.

The method may also further comprise removing the extant reagent fromthe fluid after (iv). Indeed, it is also preferred that at least (ii) to(vi) are repeated to determine further components of the analyte.

The analyte is preferably a nucleic acid template to be sequenced andthe reagent is a known type of nucleotide. It is preferred, therefore,that the ion concentration changes depending on whether the nucleotideis inserted onto the nucleic acid template.

The change in ion concentration ideally correlate to the number ofnucleotide bases inserted onto the nucleic acid template.

The encoder may provide a 2-bit binary code representing the type ofnucleotide inserted.

According to a third aspect of the invention there is provided a methodof measuring ion concentration in a buffered fluid, the methodcomprising the steps of: (i) monitoring an electrical output signal froman ISFET exposed to the fluid; (ii) releasing or adsorbing a chemicalfrom a titration electrode to the fluid to change said ion concentrationuntil the output signal reaches a predetermined threshold; and (iii)determining the quantity of chemical released or adsorbed.

The method may determine the initial ion concentration from knowledge ofthe buffer capacity and amount of chemical released or adsorbed. Themethod may, therefore, further comprise (iv) determining the initial ionconcentration from knowledge of the buffer capacity and amount ofchemical released or adsorbed. It may also further comprise repeatingparts (ii) and (iii) at two different times or with different fluids andthen:

-   -   (iv) determining the difference in initial ion concentration        from knowledge of the difference in amount of chemical released        or adsorbed in each part (ii), wherein the buffer capacity        before each part (ii) is substantially the same.

The method may repeat steps (ii) and (iii) at two different times orwith different fluids and then (iv) determine the difference in initialion concentration from knowledge of the difference in amount of chemicalreleased or adsorbed in each step (ii), wherein the buffer capacitybefore each step (ii) is substantially the same. The method may alsocomprise undoing the effects of step (ii) by adsorbing to or releasingfrom the titration electrode a substantially equal quantity of saidchemical from or to the fluid.

Thus, the method may preferably further comprise undoing or partiallyreversing the effects of part (ii) by adsorbing to or releasing from thetitration electrode a substantially equal quantity of said chemical fromor to the fluid. The method may, therefore, also preferably furthercomprise undoing or partially reversing the effects of part (ii) byadsorbing to or releasing from a second titration electrode asubstantially equal quantity of a second chemical from or to the fluid.

It is preferred that the period of part (ii) is greater than 2 seconds,preferably greater than 5 seconds, 10 seconds, or 30 seconds.

It is also preferred that the period of part (ii) is less than 200seconds, preferably less than 100 seconds, 60 seconds, or 50 seconds.

A relationship between said output signal and ion concentration ispreferably known.

Preferably, the threshold is one of: a predetermined change in theoutput signal; a predetermined rate of change in the output signal; or astate of the output signal changing from ON to OFF or vice versa.

The ion concentration changes are preferably due to a chemical reaction.The amount of chemical released or absorbed is preferably determined asthe total electric charge provided by a controller to the titrationelectrode.

The invention provides a simplified architecture for processing an ISFETsignal, which increases the integration ability of sensor and processinginto a single chip. The inherent analogue to digital conversion ofpreferred embodiments not only removes the need for several steps forsignal conditioning and analogue processing but also reduces electricalnoise associated with these steps compared to prior art devices.

Thus, in a further aspect, there is provided an array of apparatusesdiscussed above. The array may comprise a multiplexer connected to eachdevice to select which first output signal is to be demodulated.Preferably, the array is of apparatuses wherein said transducercomprises a first transistor of a first type connected to a secondtransistor of a second type to form a CMOS inverter. Preferably, saidarray comprises a multiplexer connected to each said second transistorto activate or deactivate individual devices. The array may furthercomprise a CDMA processor to encode output signal from each device,preferably enabling said output signals to be transmitted on a singlechannel.

BRIEF DESCRIPTION OF THE DRAWINGS

Specific embodiments of the invention will now be described by way ofexample only with reference to the accompanying figures, in which:

FIG. 1 is a block diagram of a system of a preferred embodiment;

FIG. 2 shows four schematic arrangements of transducers;

FIG. 3 is a schematic of a readout circuit;

FIG. 4 shows graphs of output signals of nucleic acid reactions; and

FIG. 5. shows graphs of input and output signals for a system having anoscillating chemical input.

DETAILED DESCRIPTION

In a system comprising the invention there will typically be asemiconductor substrate housing the sensor and signal processing,overlaid by a substrate containing fluidic channels and one or morechambers. The chambers contain a fluid which has an ion concentration tobe measured. The ion concentration may be constant or may be changing.The change may be a result of a chemical reaction releasing or absorbingthe ions. The limit of detection of such a system is governed by a) theamount of buffer in the fluid which masks changes in ion concentrationand b) the electrical characteristics of the transistor which requiresthe ionic charges to create an electric field to permit electric currentto flow and be detected. These can be seen as thresholds to be exceeded.In the present invention a controlled oscillating signal is provided tothe system, which in combination with the chemical/ionic signal exceedsone or both of these thresholds to provide a detectable output signal.The effect of the controlled signal is subtracted from the output signalto determine the effect of the chemical signal contribution. Byoscillating the controlled signal the net effect over time to the systemis zero. The oscillating signal is ideally continuously varying over aportion of the period such that the point at which the threshold iscrossed can be determined.

In one embodiment, the oscillating signal is a chemical provided to thefluid to change the ion concentration within the buffer. In anotherembodiment, the oscillating signal is an electrical signal provided tothe sensor transducer to change the electrical operating point.Combinations of these embodiments are envisaged and within the scope ofthe invention.

In such a system the chemical signal can be regarded as modulating theoutput signal and doing so in an efficient form for data processing.Ideally the chemical concentration is converted to a digital signal tobe further processed. Processing of this signal may be performed inhardware or software.

Electrical Oscillation

A block diagram of an exemplary system is shown in FIG. 1. A fluidsample, which contains a concentration of an analyte (including zeroconcentration where the analyte is absent from the sample), is incontact with a chemical sensing surface of an ISFET that converts theconcentration of the analyte into an analogue electrical signal. Atransducer converts the chemical signal into an analogue signal. Thetransducer is connected to an oscillating signal input. The chemicalsignal and oscillating signal combine to produce an oscillating outputsignal. If the transducer comprises an ISFET forming part of a CMOSinverter, the transducer will turn on and off to produce a pulsed outputsignal as shown. The analogue chemical signal contributes to modulatethe output signal such that the phase shifts or pulse width changes.This change represents the chemical concentration. Then a Time toDigital Converter or Phase Demodulator decodes the pulse signal andoutputs a digital signal. The contribution from the oscillating inputsignal can be deducted to leave the net chemical contribution. Thesesignals may be pre-processed and stored in memory. The Time to DigitalConverter may be of the types described in:

-   Jianjun Yu et al, 12-Bit Vernier Ring Time-to-Digital Converter in    0.13 um CMOS Technology, IEEE JOURNAL OF SOLID-STATE CIRCUITS, VOL.    45, NO. 4, April 2010; or-   Gordon W. Roberts, A Brief Introduction to Time-to-Digital    Digital-to-Time Converters, IEEE TRANSACTIONS ON CIRCUITS AND    SYSTEMS II: EXPRESS BRIEFS, VOL. 57, NO. 3, March 2010.

As discussed further below the signals may represent a series ofnucleotide incorporation reaction of a fragmented DNA strand, in whichcase the signals are further processed to sequence the complete DNA bymapping algorithm units.

In one embodiment, depicted in FIG. 2 a, a chemical sensing layer 23 iscoupled to a floating gate 20 shared by two transistors (24 and 25)arranged as an inverter. An output 22 is connected to the drains. Thevoltage on the gate will have the effect of turning one transistor onand the other off such that the output is inherently digital. Forfurther reference on this arrangement and operation see applicationPCT/IB2011/002376. Further device approaches can be extended to 2(b) or2(c) using bulk input modulation or 2(d) using pseudo CMOS logic.

By connecting a signal generator to a terminal of a transistor, thetransistor can be biased such that the output is of a predetermined formto enable a chemical signal to modulate the output as desired. Theoscillating signal may be provided by a signal generator in thesemiconductor substrate capacitively coupled to the gate of atransistor. For example an oscillating signal from the signal generatorcould electrically bias the transistor to turn on and off for setperiods, with any chemical signal adding to the bias to modulate thoseperiods. Typically the voltage of the oscillation will vary continuallybetween Vss and Vdd. For example the signal may vary from 0V to 3.3V.Sawtooth, sinusoidal, or triangular waveforms are preferred. Thefrequency of the oscillating signal is dictated by factors such as thefrequency scanning the array, the time interval of a chemical reactionbeing observed, and the resolution required for the digital outputsignal to detect a change in the ion concentration.

The ISFET inverter can be driven by common or individual signal at aspecific frequency, and the output signal phase will be modulated by thehydrogen or other target ion concentration. The output signal, whichessentially becomes a chemical modulated digital signal, dramaticallysimplifies the readout and processing blocks, and is immune to mostelectrical noise.

The number of transducers and chambers will depend on the applicationbut in preferred embodiments there will be an array of more than 10,more than 100, more than 1000, more than 10,000, more than 100,000, ormore than a 1,000,000. In applications requiring more transducers andthe chamber size will typically be smaller. In preferred embodiments,the chamber volume is less than 1 mL, less than 10 uL, less than 100 nL,less than 30 nL, less than 1 nL, or less than 100 pL.

A Code division multiple access (CDMA) technique may also beincorporated in the system to encode the data from each transducer pixelin an array of transducers, allowing all the pixel data to be sent downa single channel.

Due to its simplified readout system, methods and devices of theinvention can be widely implemented in different technologies, such as adiscrete glass ISFET or a CMOS based ISFET. For large scale integrationpurposes, CMOS based ISFETs are preferable and are described below tofurther explain the implementation.

In contrast to the embodiment of FIG. 2( a), the oscillating signal maybe connected to the bulk as shown in FIG. 2( b). FIG. 2( c) shows athird embodiment for a row of devices wherein the chemical sensingsurface is coupled to a single FET and the second FET of the inverter isswitched on and off by a row-select signal 26. For a two-dimensionalarray of transducers, the embodiment shown in FIG. 2( d) is desirable.

In FIG. 2( d) a single pixel comprises of a sensing transistor, a passtransistor, and a selection transistor. The selection transistor can beused to control the final output 22 in a single row or column. Either ofthe row select control 26 or the column select control 27 can beconnected to the oscillating signal. Since the output signal will bedigital, the transistor size can be further scaled down to sub-micron,and limited by the chemical setup only. In any of these configurations,the signal loss and distortion is negligible since a digital output isused.

All the transducer pixels can share one buffer and one front-endreadout. However, in order to further speed up the processing,transducers in one row (or column) can share one readout channel. Ineach readout channel, the digital pulses are buffered using a digitalinverter (or bit buffer_. FIG. 3 shows a counter system to readout thephase-modulated pulse signal and output a digital sequence.

The output of a time to digital converter will be truncated to a shortlength representing the change of chemical signal. This configurationcan reduce the data handled and memory required. For instance, theentire output, which might normally have 12-bits resolution, will bereduced to 4 bits and stored into memory. The base line will be treatedas common signal and stored as well. Therefore for each pixel, themaximum data required is the base count times 4 bits plus 10 bits commonmode signal.

Application to Nucleic Acid Detection and Identification

In the field of genetic testing, it is desirable to identify one or morenucleotides of a nucleic acid (such as DNA and RNA). Typically a singlestrand of nucleic acid is annealed with a probe up to or including apoint on the nucleic acid to be identified. Nucleotides will becomeincorporated to the 3′ end of the probe to extend the chain. Thisincorporation reaction has been shown to release hydrogen ions which aredetectable by an ISFET with a suitably treated sensing surface. Forexample the surface may be Silicon Nitride, Silicon Dioxide, TantalumOxide, or others shown to have sensitivity to hydrogen ions.

A nucleotide will only become incorporated if it is complementary to thenucleotide opposite. By correlating the known chemical compound added(e.g. the type of nucleotide dATP, dNTP, dTTP, dGTP, or allele specificprobe) to a change (or lack thereof) in output signal, a nucleotide at apoint of interest on the nucleic acid can be identified. Details of suchapplication and implementation have been described in patent applicationU.S. Ser. No. 11/625,844 and patents U.S. Pat. No. 7,686,929 and U.S.Pat. No. 7,888,015, incorporated herein by reference.

Sequencing

As an extension to the identification of a single nucleotide base in anucleic acid, it is desirable to identify a sequence of tens to hundredsof nucleotide bases.

In one method known as the shooting gun method, a whole chain of DNA iscut into small pieces, and copied to increase detection redundancy. Thepieces are divided amongst an array of sensor pixels. Therefore everypixel data represents a copy of a small piece of DNA. Identifying theoverlap of base sequences, in other words data similarity in memory,provides the entire DNA base sequence. This comparison can beimplemented by digital logic, i.e. XOR, AND, NOR, NAND etc. A digitalprocessing block can concatenate the sequence based on the comparisonresults. The processing can be performed in parallel with the sensingfunction, with the results of mapping feedback into the pre-processpart, to reduce the computation complexity and memory requirement.

Results

Simulation results based on a 6-base long DNA is shown in FIG. 4,wherein the graphs show (a) the base calling order and base extension ateach of four sensors over the entire reaction period (16 seconds) and(b) the oscillating reference signal and modulated output signals foreach sensor over a single reaction (2 seconds).

An exemplary simulation of a method of sequencing is described below andshown in FIG. 4. The set up parameters and outputs are given in thetable below showing the simplified results of the processing. A nucleicacid to be identified (TGACCC) is copied and cut up to provide fourfragments (Fragment 1, 2, 3, 4) with one fragment placed in one chamber,each chamber having one sensor (Sensor 1, 2, 3, 4). There would normallybe millions of identical copies of a given fragment in a chamber. Aprobe is attached to each fragment up to but not including the basesshown. The probe is not shown for simplicity of understanding.

The order of nucleotides added (the bases call order) is dATP, dCTP,dTTP, and dGTP (A, C, T, G), which is repeated until each fragment isfully extended. The fluid is initially set to pH7 and is reset to pH7after every base is added.

Every 2 seconds in this case, a new base will be added to each chamberin the order given above. Extension will occur if the added base iscomplementary to the fragment at the base opposite to release hydrogenions. For each base extension, the released hydrogen will induce aspecific pH drop, in our case, −0.2 pH per base extended. This pH willbe directly reflected by the output signal with a modulated width orphase correlating to the pH change (FIG. 4( b)). Note that in the casewhere an inverter is used, more base extension, leading to greater ionrelease, reduces the ON period of the output signal.

The oscillating input signal to the sensor is a triangular wave as shownin FIG. 4( b), having a frequency higher than the rate of base addition,preferably higher than the rate of the smallest change to be detected.

The amount of pH change can be extracted from this signal by removingthe initial pulse width (i.e. at pH 7). This is converted to the numberof bases that were extended by dividing the pH change by the rate ofchange per base (0.2 pH/base in our case). See the Sensor output in FIG.4( a). In reality the pH change is not linear for each base extended noris the change instantaneous and stable over the reaction.

By correlating the addition of the known base with the count ofextension, the sequence of each fragment can be identified. The base ofeach fragment can be represented by a 2-bit binary code provided by anencoder (e.g. 00=A, 01=T, 10=G, 11=C). Finally the identified fragmentsare reassembled using known mapping techniques (not shown):

Nucleic Acid T G A C C C Template BASE Call A C T G Repeat . . . OrderFragment 1 T G A C Fragment 2 G A C C Fragment 3 A C C C Fragment 4 G AC C Exten- Sensor1 0 1 1 0 1 0 0 1 sion Count Sensor2 0 0 1 0 1 0 0 2Sensor3 1 0 0 3 0 0 0 0 Sensor4 0 0 1 0 1 0 0 2

For example, during a 4 second period of base addition, the pH changefrom an extension reaction may last 1 second. To measure the change,capturing the peak and width of the pH change, the pH is sampled 10times over the 1 second period. The width of the ON periods will varyover the 4 second reaction window. The quantification of base extensionmay involve a complex algorithm looking at the minimum pulse width,average pulse width, sum of pulse widths, etc.

It can be seen that the output sequence can be easily derived from thisscheme.

Advantageous of preferred embodiments of a system are:

-   -   by using a novel configuration of ISFETs, the total transistor        count for a single detection pixel can be reduced to two        (including selection transistor).    -   no analogue processing circuitry is necessary.

Output is immune to conversion time and most electrical noise induced bythe digital processing circuit.

Transistor size can be reduced further since digital logic is adoptedand transistor mismatch can be neglected.

The processing and memory blocks can be integrated into a single chip.

Since all detection is synchronized, sequencing and comparison can betriggered accurately for all the pixels. Sequencing can be performedconcurrently with the reaction, dramatically reducing the processingtime and improving the processing accuracy.

In one embodiment, the reference signal is reset before each nucleotideinsertion step to compensate for any background ion concentration (forexample the concentration remaining from the previous insertion step).The reset reference signal together with the background chemical signalthus produces a consistent pre-insertion reference output signal, anysubsequent modulation to the output signal representing theconcentration change due to the current nucleotide insertion step.

In addition to sequencing a nucleic acid, the device may be used toidentify other chemicals. For example, a complex molecule having severalidentifiable elements of varying types can be identified be using astep-wise (or repeated) reaction with analyte specific reagents. Thedigital output will identify the exact type of the molecule.

Chemical Oscillation

As discussed above, in one embodiment the chemical input to the systemmay be varied by a controlled chemical signal such that the initial ionconcentration acts to modulate the output signal. The combined chemicalsignal can be swept through a range to examine the a) ion concentrationwithin the buffer and b) examine the electrical operating point of thetransistor (sub-threshold, linear, saturation). The former is usefulwhen the initial ion concentration is within the buffer capacity suchthat any small changes will be masked by the buffer. The latter may beoperated as described above with an inverter to create an output pulsewhose width is modulated by the initial ion concentration. The twoeffects may be combined such that adding titrant moves the ionconcentration operating point beyond the buffer capacity and alsocrosses a switching point of the inverter.

The effect is demonstrated in FIG. 5. In graph 5(a) a controlledquantity of titrant is added to the fluid over a first period and theremoved over a second period. The net chemical effect on the system isthus zero. In graph 5(b) the pH of two cases are plotted over the sameperiods. In the first case (solid line), the hydrogen ion concentrationis small compared to the buffer capacity and so takes a long time beforethere is a detectable pH change. In the second case (dashed line), thehydrogen ion concentration is large compared to the buffer capacity andso takes less time before there is a detectable pH change. As the ionconcentration exceeds the buffer capacity the rate of pH change perquantity of titrant added increases. This is seen as a steeper slopeafter the horizontal buffer lines.

The initial ion concentration can be measured by the time (amount oftitrant added) until the slope reaches a threshold slope or the pHchange exceeds a threshold. This is clearly an inverse relationshipbetween time and concentration. The relationship may be determined by amodel of the chemical system or determined empirically. The relationshipmay be stored as a look-up table in memory.

The pH in graph 5(b) could be used to produce an analogue output signalfrom the ISFET to be detected and measured. It is also possible to forman ion sensitive switch as described above, where two FETs are arrangedas an inverter biased to switch when the ion concentration exceeds athreshold (identified as the upper horizontal line in FIG. 5( b). Theoutput will thus be ON or OFF, wherein the period ON depends on theinitial ion concentration as shown in FIG. 5( c).

The titration reaction is naturally slow and so the oscillation periodin preferred embodiments is greater than 2 seconds, preferably greaterthan 5 seconds, 10 seconds, or 30 seconds. However, the period shouldnot be so long as to miss changes in the ion concentration or slow downdetection time. Thus in preferred embodiments, the oscillation period isless than 200 seconds, preferably less than 100 seconds, 60 seconds, or50 seconds.

A threshold may be reached when the concentration of titrated ions (T)plus initial ions (I) is greater than the buffer capacity (B).

In cases where a change in ion concentration is to be detected, theinitial ion concentration is determined at a first time using the stepsdiscussed above and illustrated in FIG. 5. The steps are repeated at asecond time to determine if more or less titrant was needed to reach thethreshold.

The threshold is reached at time 1 when:

T1+I1>B;

The threshold is reached at time 2 when:

-   -   T2+I2>B; and thus (regardless of the buffer capacity (which may        be unknown)):

(I1−I2)=ΔI=T2−T1

Thus the change in initial ion concentration is known from the amount oftitration required at each time to reach the threshold.

In cases where the initial ion concentration is to be measured onlyonce, titration may be unidirectional, such that titrant is added orremoved in a first period, but the effect is not reversed in a secondperiod. To measure the absolute initial ion concentration, it isnecessary to know the buffer capacity of the fluid and the amount oftitrant added to exceed the capacity to reach a threshold (e.g. slope orchange):

i.e. B−T=I.

In either case, the titration electrode may be operated to add orrelease chemicals, which chemicals affect the ion concentration to bedetected. The effect may be to increase or decrease ion concentrationfrom the initial concentration.

The titrant added need not be a predetermined amount, but may rather beadded until one of the thresholds is reached. In this case the amountadded is calculated.

Titration may be achieved by exposing a titration electrode to thefluid. The electrode is connected to a controller which provides acontrolled amount of electric current. Calculating the integral ofcurrent over time results in a known quantity of charge, which isproportional to the quantity of titrant released or adsorbed to theelectrode. The titration electrode may be of the type described in B.van der Schoot et al., Titration-on-a-chip, chemical sensor-actuatorsystems from idea to commercial product, Sensors and Actuators B 105(2005) 88-95.

The titration reaction at an electrode with a negative charge may byexpressed as:

2H₂O+e ⁻→H₂(gas)+2OH⁻ which has the effect of raising pH.

The titration reaction at an electrode with a positive charge may byexpressed as:

2H₂O−e ⁻→O₂(gas)+4H⁺ which has the effect of lowering pH.

Although the invention has been described in terms of preferredembodiments as set forth above, it should be understood that theseembodiments are illustrative only and that the claims are not limited tothose embodiments. Those skilled in the art will be able to makemodifications and alternatives in view of the disclosure which arecontemplated as falling within the scope of the appended claims. Forexample, references to connections may be made directly or indirectly,as appropriate. Each feature disclosed or illustrated in the presentspecification may be incorporated in the invention, whether alone or inany appropriate combination with any other feature disclosed orillustrated herein.

1. An apparatus comprising: a transducer for providing a firstelectrical output signal and arranged to receive an electrical inputsignal, the transducer switching the first electrical output signalbetween an ON and OFF state; a chemical sensing surface coupled to thetransducer for receiving a chemical input signal; and a signal generatorfor oscillating one or more of said input signals to vary the switchingpoint of the transducer.
 2. An apparatus according to claim 1, whereinthe signal generator comprises a titration electrode arranged to releaseor adsorb a chemical, which chemical affects a concentration of ionsdetected by the chemical sensing surface.
 3. An apparatus according toclaim 2, wherein the signal generator further comprises a controller toprovide an oscillating electrical current to the titration electrode.4.-38. (canceled)
 39. An apparatus according to claim 1, wherein thesignal generator oscillates the chemical input signal to switch thefirst output signal ON for a first period and OFF for a second period.40. An apparatus according to claim 1, wherein the signal generatoroscillates the electrical input signal to switch the first electricaloutput signal ON for a first period and OFF for a second period; furtherwherein the chemical input signal modulates the period of the first andsecond period.
 41. An apparatus according to claim 1, wherein the signalgenerator provides an oscillating electrical input signal to atransistor comprised in the transducer at one of: a gate, a source, adrain, or a bulk.
 42. An apparatus according to claim 1, wherein saidtransducer comprises a first transistor of a first type connected to asecond transistor of a second type to form a CMOS inverter.
 43. Anapparatus according to claim 1, wherein the first electrical outputsignal is provided to one or more further transistors to form a logiccircuit.
 44. An apparatus according to claim 1, further comprising ademodulator receiving the first electrical output signal, thedemodulator providing a second digital output signal representing themagnitude of the chemical input signal.
 45. An apparatus according toclaim 44, further comprising an encoder connected to the second outputsignal, the encoder arranged to provide a third output signal comprisinga 2-bit binary code.
 46. An apparatus according to claim 1, wherein anarray of apparatuses is formed and a multiplexer is connected to eachdevice in the array to select which first output signal is to bedemodulated.
 47. A method of determining one or more components of ananalyte in a fluid, and comprising: (i) providing the fluid to achemical transducer; (ii) oscillating one or more input signals to thetransducer to provide a modulated first output signal; (iii) combiningan analyte specific reagent with the fluid, whereby ions are produced ifthe reagent reacts with the analyte; (iv) oscillating one or more inputsignals to the transducer to provide a modulated second output signal;(v) comparing the first and second modulated second output signals toquantify a change in ion concentration of the fluid; and (vi) comparingthe change in ion concentration with a threshold to determine whetherthe reagent reacted with the analyte to determine a component of theanalyte.
 48. A method according to claim 47 further comprisingdemodulating the first and second modulated output signals to providefirst and second digital output signals representing an ionconcentration and then comparing the first and second digital outputsignals to quantify a change in ion concentration of the fluid.
 49. Amethod according to claim 47, further comprising removing the extantreagent from the fluid after (iv).
 50. A method according to claim 47,further comprising repeating (ii) to (vi) to determine furthercomponents of the analyte.
 51. A method according to claim 47, whereinthe analyte is a nucleic acid template to be sequenced and the reagentis a known type of nucleotide, and wherein the ion concentration changesdepending on whether the nucleotide is inserted onto the nucleic acidtemplate.
 52. A method according to claim 51, wherein the change in ionconcentration correlates to the number of nucleotide bases inserted ontothe nucleic acid template.
 53. A method according to claim 51, whereinthe encoder provides a 2-bit binary code representing the type ofnucleotide inserted.
 54. A method of measuring ion concentration in abuffered fluid, the method comprising: (i) monitoring an electricaloutput signal from an BEET exposed to the fluid; (ii) releasing oradsorbing a chemical from a titration electrode to the fluid to changesaid ion concentration until the output signal reaches a predeterminedthreshold; and (iii) when that predetermined threshold is reached,determining the quantity of chemical released or adsorbed.
 55. A methodaccording to claim 54, further comprising (iv) determining the initialion concentration from knowledge of the buffer capacity and amount ofchemical released or adsorbed.
 56. A method according to claim 54,further comprising repeating parts (ii) and (iii) at two different timesor with different fluids and then: (iv) determining the difference ininitial ion concentration from knowledge of the difference in amount ofchemical released or adsorbed in each part (ii), wherein the buffercapacity before each part (ii) is substantially the same.
 57. A methodaccording to claim 56, further comprising (v) undoing the effects ofpart (ii) by adsorbing to or releasing from the titration electrode asubstantially equal quantity of said chemical from or to the fluid. 58.A method according to claim 54, wherein the threshold is selected fromthe group consisting of: a predetermined change in the output signal; apredetermined rate of change in the output signal; or a state of theoutput signal changing from ON to OFF or vice versa.
 59. A methodaccording to claim 54, wherein the ion concentration changes due to achemical reaction.